PCR methods for the detection of Borrelia burgdorferi
PCR is now a standard approach for direct detection of B. burgdorferi infection in tick vectors, host reservoirs, experimentally infected laboratory animals, and clinical specimens. PCR methods that are available for detection of B. burgdorferi DNA in various specimens include standard PCR, nested PCR, competitive PCR and real-time PCR (Wang et al., 2010).
The efficiency of a PCR assay is determined by several factors. Among these, the selection of an appropriate gene target and primer set for PCR amplification are the most important. In general, a PCR primer set yielding an amplicon of 100-300 bp is recommended, as it has high amplification efficiency under standard PCR conditions and can attentuate the effects of DNA fragmentation during sample processing.
Although PCR assays targeting numerous B. burgdorferi genes have been employed extensively in research settings, only a few of these genes have been widely utilized for PCR-based detection of B. burgdorferi s. l. These include the chromosomally encoded genes rrs, flaB, recA and p66, and the plasmid-encoded gene ospA (Wang et al., 2010).
Given that the number of spirochetes in infected tissues or body fluids of patients is generally very low, appropriate procedures for sample collection, transport and DNA preparation from clinical samples are critical for yielding reliable and consistent PCR results. Sensitivity of PCR assays may be reduced by degradation of the B. burgdorferi DNA during sample transport, storage, and processing. If the tissue is kept in BSK medium for over 24 h, some spirochetes may have migrated from the tissue to the culture medium. In this case, DNA prepared from both the tissue and the medium will increase the positivity rate of the PCR assay (Wang et al., 2010).
Clinical specimens collected from patients should be subjected to DNA extraction and PCR analysis shortly after collection; if this is not possible, specimens should be kept frozen until analysis. Studies on infected animal tissues suggest that PCR with DNA prepared from fresh frozen tissues has higher yields than that from paraffin-embedded, formalin-fixed tissues. As host DNA can interfere with PCR detection of B. burgdorferi in clinical and tick samples, an optimized DNA extraction procedure is essential to yield reliable PCR results for certain samples. Also, PCR inhibitors may be present in various biological materials (blood, urine, synovial fluid and cerebrospinal fluid) obtained from patients.
Real-time PCR has been used for detection, quantification and genotyping of pathogenic B. burgdorferi species in ticks and patients (Wang et al., 2010). The sensitivity of DNA extraction using manual or automated nucleic acid preparation systems is comparable for a variety of specimen types. Only a few genes have been used as targets in real-time PCR (e.g., ospA, rrs, hbb, recA) and these assays usually have used smaller fragments (100-200 bp) than those targeted in standard PCR tests. The sensitivity, specificity and reproducibility of real-time PCR and real-time quantitative PCR (qPCR) can be affected by a number of factors, including the properties of selected primers or probe, the quality of template DNA, the detection format (SYBR Green I-based or hybridization probe-based), and the amplification conditions (Wang et al., 2010).
References:
Wang, G., Aguero-Rosenfeld, M.E., Wormser, G.P. and Schwartz, I. (2010) Detection of Borrelia burgdorferi. In: Borrelia: Molecular Biology, Host Interaction and Pathogenesis (D. Scott Samuels and Justin D. Radolf, eds.). Caister Academic Press, Norfolk, UK.
Mackay, I.M. (2007) Real-Time PCR in Microbiology: From Diagnosis to Characterization. Caister Academic Press, Norfolk, UK.
The efficiency of a PCR assay is determined by several factors. Among these, the selection of an appropriate gene target and primer set for PCR amplification are the most important. In general, a PCR primer set yielding an amplicon of 100-300 bp is recommended, as it has high amplification efficiency under standard PCR conditions and can attentuate the effects of DNA fragmentation during sample processing.
Although PCR assays targeting numerous B. burgdorferi genes have been employed extensively in research settings, only a few of these genes have been widely utilized for PCR-based detection of B. burgdorferi s. l. These include the chromosomally encoded genes rrs, flaB, recA and p66, and the plasmid-encoded gene ospA (Wang et al., 2010).
Given that the number of spirochetes in infected tissues or body fluids of patients is generally very low, appropriate procedures for sample collection, transport and DNA preparation from clinical samples are critical for yielding reliable and consistent PCR results. Sensitivity of PCR assays may be reduced by degradation of the B. burgdorferi DNA during sample transport, storage, and processing. If the tissue is kept in BSK medium for over 24 h, some spirochetes may have migrated from the tissue to the culture medium. In this case, DNA prepared from both the tissue and the medium will increase the positivity rate of the PCR assay (Wang et al., 2010).
Clinical specimens collected from patients should be subjected to DNA extraction and PCR analysis shortly after collection; if this is not possible, specimens should be kept frozen until analysis. Studies on infected animal tissues suggest that PCR with DNA prepared from fresh frozen tissues has higher yields than that from paraffin-embedded, formalin-fixed tissues. As host DNA can interfere with PCR detection of B. burgdorferi in clinical and tick samples, an optimized DNA extraction procedure is essential to yield reliable PCR results for certain samples. Also, PCR inhibitors may be present in various biological materials (blood, urine, synovial fluid and cerebrospinal fluid) obtained from patients.
Real-time PCR has been used for detection, quantification and genotyping of pathogenic B. burgdorferi species in ticks and patients (Wang et al., 2010). The sensitivity of DNA extraction using manual or automated nucleic acid preparation systems is comparable for a variety of specimen types. Only a few genes have been used as targets in real-time PCR (e.g., ospA, rrs, hbb, recA) and these assays usually have used smaller fragments (100-200 bp) than those targeted in standard PCR tests. The sensitivity, specificity and reproducibility of real-time PCR and real-time quantitative PCR (qPCR) can be affected by a number of factors, including the properties of selected primers or probe, the quality of template DNA, the detection format (SYBR Green I-based or hybridization probe-based), and the amplification conditions (Wang et al., 2010).
References:
Wang, G., Aguero-Rosenfeld, M.E., Wormser, G.P. and Schwartz, I. (2010) Detection of Borrelia burgdorferi. In: Borrelia: Molecular Biology, Host Interaction and Pathogenesis (D. Scott Samuels and Justin D. Radolf, eds.). Caister Academic Press, Norfolk, UK.
Mackay, I.M. (2007) Real-Time PCR in Microbiology: From Diagnosis to Characterization. Caister Academic Press, Norfolk, UK.
Labels: B. burgdorferi, Borrelia, Lyme disease
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